Dengue virus (DEN) is a flavivirus causing dengue fever, an acute febrile illness which occurs with increasingly high incidence in the tropics and subtropics worldwide. The 4 DEN serotypes and other flaviviruses commonly cocirculate in endemic regions, which together with their extensive antigenic cross reactivity confound serodiagnosis. Because severe outcomes (DHF/DSS) may result from previous exposure to a different DEN serotype, DEN serotype identification is important in indicating patient treatment strategies and in predicting DHF/DSS outbreaks. Commercially available diagnostic tests rely on whole virus antigens which are only flavivirus-specific. Methods will be developed for commercial production of recombinant DEN antigens (rDEN) specific for each DEN serotype using plasmids encoding truncated DEN envelope proteins fused with E. coli MBP. Bench-scale purification efforts will define affinity chromatography conditions which yield unaggregated recombinant proteins; scale up to 30-liter production lots will follow. Antigens will be evaluated for specificity and avidity by Western blotting, dot EIA, and microplate ELISA with human sera and mouse antibodies representing a range of specificities and titers determined by IFA, ELISA, and neutralization tests. Stability tests will be initiated. Incorporation of rDEN into our commercial ELISA tests will provide needed specificity and avoid laborious and hazardous virus production. PROPOSED COMMERCIAL APPLICATIONS: Dengue is a major public health threat, with over 100 million cases estimated per year, and its geographic range is expanding. Serologic tests, widely used as diagnostic aids, rely on relatively nonspecific whole virus antigens. Replacement of these with recombinant antigens is proposed, this will significantly enhance detection of dengue by contributing specificity. PanBio InDx already distributes dengue ELISA kits in over 40 countries and the market is expected to expand. The improved ELISA tests should receive wide acceptability.